Fig. 2. Compound 5 demonstrates negative cooperativity with cyclin A.

a An intramolecular FRET assay was developed for CDK2 to track the position of the activation loop. b Compound 5 has lower affinity for CDK2 in the FRET assay as cyclin concentration increases. c As cyclin concentration increases in an activity assay, the inhibitory potential of 5 decreases. Error bars represent SEM for n = 3 independent replicates. d The IC₅₀ values of compound 5 compared to ATP-site inhibitor dinaciclib with varying p-CDK2:cyclin ratios from n = 3 independent replicates. Error bars represent the 95% confidence interval of the fitted IC₅₀ values whereas the center is the best fit value. e One of two independent SPR replicates of 5 binding to CDK2 reveals a fast on, fast off binding profile. f One of two independent SPR replicates of cyclin binding to CDK2 reveals a slower off rate than that observed for small molecule inhibitors. Source data for (b–d) are provided as a Source Data file.